DNA typing is commonly used to identify the parentage of human children, and to confirm the lineage of horses, dogs, other animals, and agricultural crops. DNA typing is also commonly employed to identify the source of blood, saliva, semen, and other tissue found at a crime scenes or other sites requiring identification of human remains. DNA typing is also employed in clinical settings to determine success or failure of bone marrow transplantation and presence of particular cancerous tissues. DNA typing involves the analysis of alleles of genomic DNA with characteristics of interest, commonly referred to as “markers”. Most typing methods in use today are specifically designed to detect and analyze differences in the length and/or sequence of one or more regions of DNA markers known to appear in at least two different forms in a population. Such length and/or sequence variation is referred to as “polymorphism.” Any region (i.e. “locus”) of DNA in which such a variation occurs is referred to as a “polymorphic locus.” The methods and materials of the present invention are designed for use in the detection of multiple loci of DNA, some or all of which are polymorphic loci.
Genetic markers which are sufficiently polymorphic with respect to length or sequence have long been sought for use in identity applications, such as paternity testing and identification of tissue samples collected for forensic analysis. The discovery and development of such markers and methods for analyzing such markers have gone through several phases of development over the last several years.
The first identified DNA variant markers were simple base substitutions, i.e. simple sequence polymorphisms, which were most often detected by Southern hybridization assays. For examples of references describing the identification of such markers, designed to be used to analyze restriction endonuclease-digested DNA with radioactive probes, see: Southern, E. M. (1975), J. Mol. Biol. 98(3):503–507; Schumm, et al. (1988), American Journal of Human Genetics 42:143–159; and Wyman, A. and White, R. (1980) Proc. Natl. Acad. Sci, U.S.A. 77:6754–6758.
The next generation of markers were size variants, i.e. length polymorphisms, specifically “variable number of tandem repeat” (VNTR) markers (Nakamura Y., et al. (1987), Science 235: 1616–1622; and U.S. Pat. No. 4,963,663 issued to White et al. (1990); U.S. Pat. No. 5,411,859 continuation of 4,963,663 issued to White et al. (1995)) and “minisatellite” markers (Jeffreys et al. (1985a), Nature 314:67–73; Jeffreys et al. (1985b) Nature 316:76–79, U.S. Pat. No. 5,175,082 for an invention by Jeffreys). Both VNTR and minisatellite markers, contain regions of nearly identical sequences repeated in tandem fashion. The core repeat sequence is 10 to 70 bases in length, with shorter core repeat sequences referred to as “minisatellite” repeats and longer repeats referred to as VNTRs. Different individuals in a human population contain different numbers of the repeats. The VNTR markers are generally more highly polymorphic than base substitution polymorphisms, sometimes displaying up to forty or more alleles at a single genetic locus. However, the tedious process of restriction enzyme digestion and subsequent Southern hybridization analysis are still required to detect and analyze most such markers.
The next advance involved the joining of the polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202 by Mullis, K. B.) technology with the analysis of VNTR loci (Kasai, K. et al. (1990) Journal Forensic Science 35(5):1196–1200). Amplifiable VNTR loci were discovered, which could be detected without the need for Southern transfer. The amplified products are separated through agarose or polyacrylamide gels and detected by incorporation of radioactivity during the amplification or by post-staining with silver or ethidium bromide. However, PCR can only be used to amplify relatively small DNA segments reliably, i.e. only reliably amplifying DNA segments under 3,000 bases in length Ponce, M & Micol, L. (1992) NAR 20(3):623; Decorte R, et al. (1990) DNA Cell Biol. 9(6):461–469). Consequently, very few amplifiable VNTRs have been developed.
In recent years, the discovery and development of polymorphic short tandem repeats (STRs) as genetic markers has stimulated progress in the development of linkage maps, the identification and characterization of diseased genes, and the simplification and precision of DNA typing. Specifically, with the discovery and development of polymorphic markers containing dinucleotide repeats (Litt and Luty (1989) Am J. Hum Genet 3(4):599–605; Tautz, D (1989) NAR 17:6463–6471; Weber and May (1989) Am J Hum Genet 44:388–396; German Pat. No. DE 38 34 636 C2, inventor Tautz, D; U.S. Pat. No. 5,582,979 filed by Weber, L.), STRs with repeat units of three to four nucleotides (Edwards, A., et al. (1991) Am. J. Hum. Genet. 49: 746–756.; Hammond, H. A., et al. (1994) Am. J. Hum. Genet. 55: 175–189; Fregeau, C. J.; and Fourney, R. M. (1993) BioTechniques 15(1): 100–119.; Schumm, J. W. et al. (1994) in The Fourth International Symposium on Human Identification 1993, pp. 177–187 (pub. by Promega Corp., 1994); and U.S. Pat. No. 5,364,759 by Caskey et al.; German Pat. No. DE 38 34 636 C2 by Tautz, D.) and STRs with repeat units of five to seven bases (See, e.g. Edwards et al. (1991) Nucleic Acids Res. 19:4791; Chen et al. (1993) Genomics 15(3): 621–5; Harada et al. (1994) Am. J. Hum. Genet. 55: 175–189; Comings et al. (1995), Genomics 29(2):390–6; and Utah Marker Development Group (1995), Am. J. Genet. 57:619–628; and Jurka and Pethiyagoda (1995) J. Mol. Evol. 40:120–126)), many of the deficiencies of previous methods have been overcome. STR markers are generally shorter than VNTR markers, making them better substrates for amplification than most VNTR markers.
STR loci are similar to amplifiable VNTR loci in that the amplified alleles at each such locus may be differentiated based on length variation. Generally speaking STR loci are less polymorphic at each individual locus than VNTR loci. Thus, it is desirable to amplify and detect multiple STR systems in a single amplification reaction and separation to provide information for several loci simultaneously. Systems containing several loci are called multiplex systems and many such systems containing up to 11 separate STR loci have been described. See, e.g., Proceedings: American Academy of Forensic Sciences (Feb. 9–14, 1998), Schumm, James W. et al., p. 53, B88; Id., Gibson, Sandra D. et al., p. 53, B89; Id., Lazaruk, Katherine et al., p. 51, B83; Sparkes, R. et al., Int J Legal Med (1996) 109:186–194; AmpFlSTR Profiler™ PCR Amplification Kit User's Manual (1997), pub by Perkin-Elmer Corp, i–viii and 1—1 to 1–10; AmpFlSTR Profiler Plus™ PCR Amplification Kit User's Manual (1997), pub by Perkin-Elmer Corp., i viii and 1—1 to 1–10; AmpFlSTR COfiler™ PCR Amplification Kit User Bulletin (1998), pub by Perkin-Elmer Corp. i–iii and 1—1 to 1–10; 9th International Symposium on Human Identification (Oct. 7–10, 1998), pub. by Promega Corp., Staub, Rick W. et al., Poster Abstract 15; Id., Willard, Jeanne M. et al., Poster Abstract 73; and Id., Walsh, P. Sean, et al., Speaker Abstract for 8:50 am–9:20 am, Thursday, Oct. 8, 1998.
Amplification protocols with STR loci can be designed to produce small products, generally from 60 to 500 base pairs (bp) in length, and alleles from each locus are often contained within a range of less than 100 bp. This allows simultaneous electrophoretic analysis of several systems on the same gel or capillary electrophoresis by careful design of PCR primers such that all potential amplification products from an individual system do not overlap the range of alleles of other systems. Design of these systems is limited, in part, by the difficulty in separating multiple loci in a single gel or capillary. This occurs because there is spacial compression of fragments of different sizes, especially longer fragments in gels or capillaries, i.e., commonly used means for separation of DNA fragments by those skilled in the art.
The United States Federal Bureau of Investigation (“FBI”) has established and maintains a Combined DNA Index System (“CODIS”), a database of DNA typing information. Local, state, and national law enforcement agencies use the CODIS system to match forensic DNA evidence collected at crime scenes with DNA information in the database. CODIS and other national database systems have proven to be an effective tool for such agencies to use in solving violent crimes. (See, e.g. Niezgoda, Stephen, in Cambridge Healthtech Institute's Second Annual Conference on DNA Forensics: Science, Evidence, and Future Prospects (Nov. 17–18, 1998), pp. 1–21.; Niezgoda, Stephen in Proceedings From The Eighth International Symposium on Human Identification 1997, pub. by Promega Corporation (1998), pp 48–49; Frazier, Rachel R. E. et al. Id., pp. 56–60; Niezgoda, S. J. Profiles in DNA 1(3): 12–13; Werrett, D. J. and Sparkes, R. in Speaker Abstracts: 9th International Symposium on Human Identification (Oct. 7–10, 1998) pp. 5–6). Until recently, only restriction fragment length polymorphism (“RFLP”) data obtained from the analysis of particular VNTR loci was considered a core component in the database. The FBI has recently identified thirteen polymorphic STR loci for inclusion in the CODIS database. The thirteen CODIS STR loci are HUMCSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31. (Budowle, Bruce and Moretti, Tamyra in Speaker Abstracts: 9th International Symposium on Human Identification (Oct. 7–10, 1998) pp. 7–8). Both VNTR and STR marker data are currently maintained in the CODIS database. (See, e.g. Niezgoda, Stephen in Second Annual Conference on DNA Forensics, supra). Until the present invention, the number of loci which could be co-amplified in a single reaction, and analyzed thereafter was limited. Specifically, no materials or methods had been developed for use in multiplex amplification of thirteen or more STR loci, much less the thirteen polymorphic STR loci identified for use in the CODIS database.
The materials and methods of the present method are designed for use in multiplex analysis of particular polymorphic loci of DNA of various types, including single-stranded and double-stranded DNA from a variety of different sources. The present invention represents a significant improvement over existing technology, bringing increased power of discrimination, precision, and throughput to DNA profiling for linkage analysis, criminal justice, paternity testing, and other forensic, medical, and genetic identification applications.